Angiogenesis is a fundamental process required for normal growth and development of tissues, and involves the proliferation of new capillaries from pre-existing blood vessels. Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis which takes place in the healthy individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis and arthropathies. Inhibition of angiogenesis is useful in preventing or alleviating these pathological processes.
Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor-α (TGFα), and hepatocyte growth factor (HGF). See for example Folkman et al, “Angiogenesis”, J. Biol. Chem., 267:10931-10934, 1992, for a review.
It has been suggested that a particular family of endothelial cell-specific growth factors and their corresponding receptors is primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF/VEGF family, which act via receptor tyrosine kinases (RTKs).
Numerous PDGF/VEGF family members have been identified. These include PDGF-A (see e.g., GenBank Acc. No. X06374), PDGF-B (see e.g., GenBank Acc. No. M12783), PDGF-C (Intl. Publ. No. WO 00/18212), PDGF-D (Intl. Publ. No. WO 00/027879), VEGF (also known as VEGF-A or by particular isoform), Placenta growth factor, PlGF (U.S. Pat. No. 5,919,899), VEGF-B (also known as VEGF-related factor (VRF) Intl. Publ. No. PCT/US96/02597 and WO 96/26736; U.S. Pat. Nos. 6,331,301; 5,840,693; 5,928,939; 5,607,918), VEGF-C, (U.S. Pat. Nos. 6,645,933; 6,403,088; 6,361,946; 6,221,839; 6,130,071 and International Patent Publication No. WO 98/33917), VEGF-D (also known as c-fos-induced growth factor (FIGF) (U.S. Pat. Nos. 6,383,484 and 6,235,713, Intl. Publ. No. WO98/07832), VEGF-E (also known as NZ7 VEGF or OV NZ7; Intl. Publ. No. WO00/025805 and U.S. Patent Publ. No. 2003/0113870), NZ2 VEGF (also known as OV NZ2; see e.g., GenBank Acc. No. S67520), D1701 VEGF-like protein (see e.g., GenBank Acc. No. AF106020; Meyer et al., EMBO J. 18:363-374), and NZ10 VEGF-like protein (described in Intl. Patent Application PCT/US99/25869) [Stacker and Achen, Growth Factors 17:1-11 (1999); Neufeld et al., FASEB J 13:9-22 (1999); Ferrara, J Mol Med 77:527-543 (1999)].
The PDGF/VEGF family proteins are predominantly secreted glycoproteins that form either disulfide-linked or non-covalently bound homo- or heterodimers whose subunits are arranged in an anti-parallel manner [Stacker and Achen, Growth Factors 17:1-11 (1999); Muller et al., Structure 5:1325-1338 (1997)]. Each VEGF family member has between 30% and 45% amino acid sequence identity with VEGF. The VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cysteine knot motif. Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties.
Vascular endothelial growth factors appear to act by binding to receptor tyrosine kinases of the PDGF/VEGF-receptor family. Six endothelial cell receptor tyrosine kinases which bind PDGF/VEGF molecules have been identified, namely Flt-1 (VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4 (VEGFR-3), Tie and Tek/Tie-2, and the PDGF receptor. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction. The essential, specific role in vasculogenesis and angiogenesis of Flt-1, Flk-1, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos.
VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and placenta growth factor (PlGF). VEGF-C has been shown to be a ligand for Flt4 (VEGFR-3), and also activates VEGFR-2 (Joukov et al., EMBO J., 15: 290-298, 1996). VEGF-D binds to both VEGFR-2 and VEGFR-3. A ligand for Tek/Tie-2 has been described (International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc.); however, the ligand for Tie has not yet been identified.
VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells. Both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., Oncogene, 8: 11-18, 1992; Kaipainen et al., J. Exp. Med., 178: 2077-2088, 1993; Dumont et al., Dev. Dyn., 203:80-92, 1995; Fong et al., Dev. Dyn., 207:1-10, 1996) and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl. Acad. Sci. USA, 9: 3566-3570, 1995). VEGFR-3 is also expressed in the blood vasculature surrounding tumors.
Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation. Analysis of embryos carrying a completely inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al., Nature, 376: 66-70, 1995). However, deletion of the intracellular tyrosine kinase domain of VEGFR-1 generates viable mice with a normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95:9349-9354, 1998). The reasons underlying these differences remain to be explained but suggest that receptor signaling via the tyrosine kinase is not required for the proper function of VEGFR-1. Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoesis and vasculogenesis (Shalaby et al., Nature, 376: 62-66, 1995; Shalaby et al., Cell, 89: 981-990, 1997). Inactivation of VEGFR-3 results in cardiovascular failure due to abnormal organization of the large vessels (Dumont et al., Science 282:946-949, 1998).
VEGFR-3 is widely expressed on endothelial cells during early embryonic development but as embryogenesis proceeds becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., Cancer Res., 54:6571-6577, 1994; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92:3566-3570, 1995). VEGFR-3 is expressed on lymphatic endothelial cells in adult tissues. This receptor is essential for vascular development during embryogenesis. Abnormal development or function of the lymphatic endothelial cells can result in tumors or malformations of the lymphatic vessels, such as lymphangiomas or lymphangiectasis. Witte, et al., Regulation of Angiogenesis (eds. Goldber, I. D. & Rosen, E. M.) 65-112 (Birkä user, Basel, Switzerland, 1997). The VEGFR-3 receptor is upregulated in many types of vascular tumors, including Kaposi's sarcomas (Jussila et al., Cancer Res 58, 1955-1604, 1998; Partanen et al., Cancer 86:2406-2412, 1999). The importance of VEGFR-3 signaling for lymphangiogenesis was revealed in the genetics of familial lymphedema, a disease characterized by a hypoplasia of cutaneous lymphatic vessels, which leads to a disfiguring and disabling swelling of the extremities (Witte, et al., Regulation of Angiogenesis (supra); Rockson, S. G., Am. J. Med. 110, 288-295, 2001). Additional studies demonstrated that signaling through the VEGFR-3 receptor is sufficient to induce lymphangiogenesis (Viekkola et al., EMBO J. 20:1223-31, 2001). Further, the ligands for VEGFR-3, VEGF-C and VEGF-D, are also involved in pathogenic angiogenesis in some tumors.
Recent evidence on the association of lymphangiogenic growth factors with intralymphatic growth and metastasis of cancers (PCT/US99/23525; WO 02/060950; Mandriota, et al., EMBO J. 20:672-682, 2001); Skobe et al., Nat. Med. 7:192-198, 2001); Stacker et al., Nat. Med. 7:186-191, 2001); Karpanen et al., Cancer Res. 61:1786-1790, 2001) has provided an indication for anti-lymphangiogenic agents for tumor therapy. VEGF-C and VEGF-D signaling through the VEGFR-3 receptor has been shown to be the primary source of lymphangiogenic activation and has also been noted in pathogenic angiogenesis in some tumors.
Cancer cells spread within the body by direct invasion to surrounding tissues, spreading to body cavities, invasion into the blood vascular system (hematogenous metastasis), as well as spread via the lymphatic system (lymphatic metastasis). Regional lymph node dissemination is the first step in the metastasis of several common cancers and correlates highly with the prognosis of the disease. The lymph nodes that are involved in draining tissue fluid from the tumor area are called sentinel nodes, and diagnostic measures are in place to find these nodes and to remove them in cases of suspected metastasis. However, in spite of its clinical relevance, little is known about the mechanisms leading to metastasis via the bloodstream or via the lymphatics.
Thus, there remains a need in the art to find modulators of the growth factors and receptors involved in angiogenesis and lymphangiogenesis. Additionally, there continues to be a need for new modulators that act as specific regulators of tumor cells to improve therapy over current, non-specific cancer therapeutics, and preferably provide low, therapeutic doses and reduced toxicity and side effects to the patient.